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Derek Lowe's commentary on drug discovery and the pharma industry. An editorially independent blog from the publishers of Science Translational Medicine. All content is Derek’s own, and he does not in any way speak for his employer.

More Bacterial Targets Than You Know

Do you want to find new drugs treat human bacterial infections? (If you don’t, you’d better hope that someone else does!) The standard view in the field has long been that you have to target some essential pathway in those bacteria, one that doesn’t have a counterpart in human biology (or whose sequence and structure is different enough so that there’s plenty of therapeutic room). That makes a lot of sense, because then you’re maximizing the chances of killing off the infectious agent while minimizing the chances of human side effects. The problem is that this approach is leaving us with an increasingly limited field in which to work. The distinction in the field is between bactericidal compounds (the killers) and bacteriostatic ones (which just slow or stop growth), and in general, the feeling has been that since ars longa, vita brevis, you’re better off working on the former.

This new paper, though, a multicenter collaboration between the Broad Institute, Harvard, MGH, Chicago, and Argonne, suggests that we might be able to open up more. The authors describe an inhibitor of Mycobacterium tuberculosis tryptophan synthase, which is not an enzyme that one would normally think of targeting (it’s not essential for growth of the bacterium as long as there’s sufficient tryptophan available). To be fair, the team wasn’t targeting it – their lead compound came out of a flat-out screen of growth inhibition against one of the Broad’s chemical libraries. A lot of the time, when you try that sort of screen, you end up discovering things that have been discovered before, and a lot of them are toxic to human cells, too. But in this case, BRD4592 emerged and turned out not only to be an inhibitor of the tryptophan synthase, but to bind at an allosteric site.

That’s an interesting feature, too. Allosteric inhibition has long been on the list of “Yeah, that’s really interesting, we ought to find some way to target that more often”. For those outside the field, an allosteric inhibitor works not by binding right at a protein’s site of action, but at some other region entirely. This binding event shifts the conformation of the whole protein around to the point where its activity is affected. Allosteric sites are often part of regulatory pathways, responding to separate signaling molecules to change the activity of the underlying protein. Targeting them deliberately is not so easy; I’d guess that the great majority of allosteric inhibitors have been found by more or less random screening, in the sense of “Hmm, that doesn’t seem to bind at the active site, must be some allosteric thing”. There are probably more of them known for receptor proteins than for enzymes, but it’s a field with a lot of interest.

The tryptophan synthase enzyme is a complex one, and the authors go into detail on how BRD4592 seems to work by several different mechanisms simultaneously (there’s a crystal structure to help). It’s a weird system: the compound increases the stability of the enzyme structure, and increases its affinity for its substrate, so you’d think it would be some sort of activator. But it’s a pretty strong inhibitor of activity. It seems to both keep an indole intermediate from moving between two active sites in the protein complex, and to stabilize the enzyme/product state (which keeps the enzyme from turning over and starting a new reaction). The authors believe that it might be the most mechanistically complex allosteric inhibitor yet reported – you would not have designed this one from first principles, that’s for sure.

The enzyme, for its part, seems to be conditionally essential: it’s apparently more important during infection of host organisms than it is in the culture dish. It’s long been realized that there are such targets out there in bacteria, but they’re understandably harder to track down, since these differences only show up under specific conditions, some of which may be hard to replicate. But this means that there are compounds that are bactericidal at times and bacteriostatic at others, and since we’re going to need all the help we can get against resistant bacteria, we’d better come to grips with these things.

BRD4592 itself, though, is probably not going to be one of those compounds. As a screening hit, it’s unoptimized for the properties needed for an actual drug (and indeed, it’s not active in mice because it’s so rapidly cleared by metabolism). But that’s what drug development does – turns screening hits into drugs – and this looks like it could be a good starting point. That azetidine ring with the alpha nitrile, essential for binding, might be tricky to deal with, but a valid hit is a valid hit. Let’s see if someone can make something of this chemical series, or of the target in general.

45 comments on “More Bacterial Targets Than You Know”

Other than Saxagliptin , a DP4 inhibitor which is also amino nitrile (deactivated?), BRD4592 or its analogs might have an issue with reverse Strecker reaction, and the resultant species becomes a potent alkylating agent!

I’m not an author but helped with the logistics of the screening and data analysis. One thing mentioned in the intro that’s worth highlighting is that this compound is derived from a Diversity-Oriented Synthesis (DOS) library. We still run into people who hear DOS and think 700 Da compounds. This azetidine library has various other related cores (4-6, 4-8 ring systems) that are also showing interesting activity in a number of projects including anti-infectives. For example last year’s paper on antimalarials (where I am a coauthor) Nature 538, 344–349 highlights several related scaffolds. One series targets Pf PheRS, but the stereochemically diverse nature of the library was key in both projects as the configuration of the core is essential for activity.

Fifteen years ago, Abbott gave up on a novel macrolide antibiotic that was efficacious and safe. But it perverted the sense of taste. And they judged that–as long as there were other drugs in the niche–there was no market for one in which you could anticipate compliance problems. If there were no other drugs for the indication, of course they would have gone to market with this one.

The assertion that “you have to target some essential pathway in those bacteria, one that doesn’t have a counterpart in human biology” is false on face of it. Plenty of efficacious, safe, profitable antibiotics interfere with bacterial ribosomal function. These plainly “have a counterpart in human biology”.

True, but it’s a matter of degree here. Our ribosomes and the bacteria ones diverged about 3 billion years ago, and there’s obviously plenty of room for selectivity between the 70S and 80S models. I’ll reword a bit, though, anyway.

For aminoglycosides, the selectivity comes from uptake. They are toxic to the mammalian cell that they get into (proximal tubule, for one). AG mechanism of action is also far more complex than just inhibition of protein synthesis.

Several antibiotics on the market DO inhibit HUMAN mitochondrial protein synthesis but our cells seem to be fine for the short duration of the treatment. Can anyone make sense of that? Is the protein turnover rate in the mitochondrion abnormally slow? I would be glad to hear comments on that.

There was a paper recently with a structure of the yeast mitoRibosome that suggested that changes to the mitoribosome exit tunnel and E-site, compared to the bacterial ribosome, confers resistance to many of the ribosome targeting antibiotics in mitoribosomes. They have an example with erythromycin.

Since ribosomes are essential for life, they make attractive targets for antibiotic drugs. Of course, you need to be careful not to attack our own ribosomes, otherwise you would kill yourself along with the infection! Fortunately, bacterial ribosomes have many small differences from our own ribosomes, so there are many antibiotic drugs that specifically attack 70S ribosomes. Two examples are included here, and there are many other examples in the PDB. On the left, tetracycline (in red) is bound to the small subunit (1hnw ), blocking the binding of the mRNA. On the right, chloramphenicol (in red) is (1nji ), blocking the reaction that adds amino acids to the growing protein.”

You can download the structures of the eukaryotic ribsomes as well and compare them for yourself.

The paper is behind a pay firewall. The abstract states that the enzyme is essential in vivo but not in vitro. You stated that they stated that the compound appears to be working through several mechanisms. You also stated that it wasn’t active in mice due to metabolism. Okay. So you have to understand that without being able to read the paper (and I’m not going to that site we’re not supposed to use – I might want to run for president one day), your post raised a few questions.

1. Was this compound screened in any cytotox assay? If so, what was the safety index versus activity. TB can be cultured in PBMCs and macrophages – don’t know what they used. Did they do cytotoxic in PBMCs?
2. How do you show the essential need for this enzyme in vivo using a compound that isn’t active in vivo? A different species other than mice?
3. If there are several mechanisms as you state, how do you know which one is critical in vivo.
4. If this enzyme is so essential, why do need an allosteric inhibitor? Wouldn’t any type of inhibitor work? How about a good old fashion chemically stable substrate competitive inhibitor? Is this a suicide inhibitor and does this compound react at the allosteric site or just bind?
5. So it came out of a bacteriostatic screen but was bactericidal in vivo even though it’s metabolically (and probably chemically) unstable and it looks like something that would react with ……..well, a whole lot of things. It’s just a masked iminium species, right?

After reading the paper do this all add up? It might. But with what you wrote, I am a bit skeptical.

From what I can see:
1. The compound had a CC50 of >100 micromolar against HepG2 cells, and no activity in 36 PubChem screens.
2. I didn’t mention it, but they did show activity in a zebrafish embryo model of infection.
3. Who knows?
4. There don’t seem to be any small-molecules inhibitors known (and it’s not like they were trying for an allosteric one; that’s just what they got). There are some substrate-based inhibitors, such as indole-propanol-phosphate, but they’re much weaker.
5. Might be, although the X-ray structure seems to show the nitrile still there, and no evidence of covalency. The inhibition is reversible, without slow-binding kinetics.

Not exactly an allosteric inhibitor, but GKS put an oxaborole based compound with excellent in vitro (MIC) activity against Gram negative pathogens (including carbapenem resistant strains). The compound targeted a bacterial leucyl t-RNA synthetase editing site, which insured the correct amino acid was charged onto the correct t-RNA. It was known that in vitro selection with the compound led to a fair number of multiple single amino acid mutations in the editing site resulting in single step, high level resistance. Nonetheless, GSK decided to go into the clinic with a small Ph II study, in hopes that the in vitro resistance might not translate in man. Alas, resistance turned up very quickly, within a matter of days.

Ah – but this is MTB – where almost all drugs are single-target inhibitors and yield to single step resistance – but this is OK because treatment is with a combination of 3-4 drugs which reduce resistance selection. As is done with HIV and HCV. We need to investigate combinations of such single-targeted agents with the more standard ESKAPE pathogens.

Has anyone ever looked into using inhibitors as a preventative measure instead of antibiotics? Anythng that can give the body an edge before an infction gets going could be a way of reducing overall antibiotics use and, if we’re just a little bit lucky, extend the useful time window for new antibiotics.

A preventative inhibitor of bacterial growth would be a selective darwinian pressure for evolution of bacteria that can evade or defeat it. That’s the scenario of (many tons of) antibiotics given chronically at low doses as “growth promoters” in livestock.

Oxazolidinones are potent inhibitors of mammalian mitochondrial protein production (see McKee et al. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 2006, p. 2042–2049 or doi:10.1128/AAC.01411-05) , yet they are widely used in the clinic. Therefore, inhibition of mitochondrial protein production is not necessarily a deal breaker to arrive at new antibiotics. Still, I cannot wrap my head around this. What biological or biochemical mystery makes this possible?

Maybe the former, but as for the latter, feel free to email with anything at a similar level that you happen to find. I get a lot of good blog post ideas that way. As is the case with many of my posts, I started writing the post, and then had to go to the paper to see who’d written it and where they were from.

How long until we can target bacteria in a species dependent manner? There’s all sorts of literature out there now describing just how damaging broad acting antibiotics are for the human microbiome. It can take years, even a decade or more, for your gut bugs to fully recover fully if they ever do at all.

currently, if we want to address a bacterial infection while sparing the gut fauna, therapeutic ‘phage is probably the best tool we have. But to the best of my knowledge, our FDA hasn’t figured out how to regulate/license it. It becomes an important thing to figure out as antibiotics are ever less efficacious.

Having worked most of my adult life with phages for various molecular biology reasons, it is very, very easy to select numerous phage receptor mutants in bacteria, such that the phage no longer attaches. Most propose a cocktail of phages to sidestep this problem. For Gram negatives, a concern is rapid LPS release due to bacterial lysis which could lead to septic shock. Finally, these are very large proteins which the immune system will probably generate antibodies against, neutralizing future therapy.

That said, it would be helpful to see 2-3 well controlled clinical trials that tested phage therapy, which either has a place in medicine or isn’t efficacious enough. All I have seen are Russian anecdotal reports to date.

The great promise of ‘phage is that they mutate faster than bacteria can escape. One is therefore always administering a cocktail of genetic variants (and more are generated in vivo during treatment). There is consequently never an NCE to patent, and to test for efficacy and safety. Those relentless mutations mean that each one is in principle novel and non-obvious (and may be utile if it cures something). But a valid patent would have to enable your competitors. Would you you have to publish the ‘phage’s whole genetic sequence? Would that patent then cover the inevitable mutants?
I don’t imagine this is anything our FDA is eager to address.

“better prospects for patentability relates to genetically modified phages that are – due to human intervention – enabled to target only specific bacteria. This technology was recently presented by MIT researchers at the 2014 American Society for Microbiology Meeting. The researchers led by Timothy Lu had genetically engineered phages that use a DNA-editing system called CRISPR to target and kill only antibiotic-resistant bacteria while leaving other susceptible cells untouched. The significant engineering and alteration of natural products and processes involved in such inventions would most likely meet both the Myriad and Prometheus standards.

Yet, while the USPTO has recently issued new patent eligibility guidance and the CAFC has begun to directly apply Prometheus and Myriad to reject patent claims in biotech cases (e.g. In re Roslin), many questions remain unsolved. In particular, it is still not sufficiently clear exactly how much modification is required to render a molecule or method sufficiently distinct from naturally occurring product and processes. And even if the patent-eligibility threshold could be met in extraordinarily circumstances, the claimed invention would still have to fulfil other patentability requirements such as novelty, non-obviousness and the written description-requirements. The threshold for these requirements, however, have been heightened in recent years (see e.g. KSR v. Teleflex (2007) , Nautilus (2014) etc.). Considering that phage therapy is almost a century old with a substantial common general knowledge and a state of the art employing routine methods, these crucial requirements might still prevent the patentability of many useful applications.

Consequently, much uncertainty remains within the nowadays rather blurry lines between the “patent eligibility”, novelty, and “nonobviousness” criteria. Patents are possible, but only in narrowly defined areas since the fruits are hanging low, and the number of patients in which this technology could be applied is (so far) rather limited.

Having worked with phages too you can select for genus level specific phages by changing the isolation process. My lab pulled out hundreds of genus-wide phages without problem. The problems of using phages (except for the FDA) can be solved by isolating the right phages upfront.

This company, Adaptive Phage Therapeutics, is developing custom phage cocktails to treat life-threatening drug-resistant infections. They have sidestepped the issues of rapidly developing resistance by targeting their therapies only to people in acute need who have failed antibiotic therapy. If the therapy begins to flag due to developing resistance, they can re-screen their collection for a new cocktail. Personalized antibiotic therapy. They have already done a proof of principle eIND, but the question of scale remains.http://www.businesswire.com/news/home/20170426006880/en/Adaptive-Phage-Therapeutics—Terminally-Ill-Patient

Just eat some dirt, kiss a dog on the face, take a mega-dose of probiotics like Elixa, eat tons of fiber to feed your new bugs, and you should be fine. But if that doesn’t work, you can always go for the fecal transplant.

Allostery has long ago been shown to be what we call now as a ” sticky hit “….something in a screening deck that just adheres to whatever…a hydrophobic surface that is there…whatever. Grow up. This is the 21st century and stop reading will jencks books to understand biology.

have to agree with sentiments above by anon and the comment about why this paper is in ncb. seems to me the ELT paper from natcomm should be in ncb (from which it was likely rejected and ended up in natcomm) and this paper from broad should be somewhere else. the ELT paper is very interesting.

I asked an expert once on how many bacterial targets differed from human ones- he ran a major company research department in Boston that was Big in the 90s and sold out to an Asian company. No names please. They had a state of the art program in genomics and proteomics in bacteriology. His answer? “26”

Hmm…. Not sure that company (starting with “M” acquired by a company starting with “T”?) has had how many novel antibiotics they had discovered and developed to the market to help patients. The targets have to be validated thru the drug development as every knows.